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  • Blotting NIH3T3 Superdex200 Fractions

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    kron541
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    NIH-3T3 cells were grown, collected, and frozen into a pellet. The pellet was lysed using a lysis buffer and dounce homogenizer. The lysate was centrifuged and the cleared supernatant was collected, with a protein concentration of 13.8 mg/ml. Around 200ul of lysate was loaded onto a size exclusion chromatography column to separate proteins by molecular weight, collecting 1ml fractions that were later TCA precipitated and analyzed by SDS-PAGE.

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    NIH-3T3 cells were grown, collected, and frozen into a pellet. The pellet was lysed using a lysis buffer and dounce homogenizer. The lysate was centrifuged and the cleared supernatant was collected, with a protein concentration of 13.8 mg/ml. Around 200ul of lysate was loaded onto a size exclusion chromatography column to separate proteins by molecular weight, collecting 1ml fractions that were later TCA precipitated and analyzed by SDS-PAGE.

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    12 views1 page

    Blotting NIH3T3 Superdex200 Fractions

    Uploaded by

    kron541
    NIH-3T3 cells were grown, collected, and frozen into a pellet. The pellet was lysed using a lysis buffer and dounce homogenizer. The lysate was centrifuged and the cleared supernatant was collected, with a protein concentration of 13.8 mg/ml. Around 200ul of lysate was loaded onto a size exclusion chromatography column to separate proteins by molecular weight, collecting 1ml fractions that were later TCA precipitated and analyzed by SDS-PAGE.

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    of 1

    Blotting NIH-3T3 Gel Filtration Fractions

    Grow NIH-3T3 cells in 15cm pertri-plates and collected cells from 5 plates and
    froze them as a pellet at -80C

    Thawed the pellet on ice and resuspended them in 500ul of lysis buffer (20mM
    HEPES(pH7.2), 150mM NaCl, 2mM EDTA, 1mM DTT, 1xPiC and 1xPMSF).

    Used a small volume dounce homogenizer to lyse the cells on ice, 40x up/down
    strokes.

    The lysate was then spun at 100,000xg for 1hr @4C in a SW55.1 rotor.

    The cleared lysate was collected and the protein concentration was measured by
    Bradford’s Assay

    (Note: usually I get about 10 to 15mg/ml. Since I lyse in 0.5mls and after the
    centrifugation I only collect about 400ul, I do 2 superdex-200 runs with
    about to 2-4mgs of load per run. I have run up to 10mgs of lysate on a
    superdex-200 column.)

    The concentration of the lysate was calculated to be around 13.8mg/ml and since
    I had little more than 400ul of cleared lysate I had a total of about 5.5mg of
    lysate.

    Loaded about 200ul of lysate on the superdex-200 column and collected 1ml
    fractions

    I did two runs and TCA precipitated each 1ml fraction

    I resuspended each TCA precipitated fraction in 50ul of 1xSB

    I pooled the two 50ul resuspended TCA pellets and then ran 25ul of the pooled
    reactions on two 6% and two 15% SDS-PAGE.

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